Checkpoint inhibitors towards targets such as PD1 and PD-L1 are being developed to activate quiescent T-cells embedded in tumors and enable them to recruit the rest of the immune system to fight a cancer mass. How these new cures reactivate the relatively few Tumor-Infiltrating Lymphocytes (TILs) enabling the release of a complex message of cytokines to recruit the immune system, thus impacting treatment efficacy. Detecting these T-cells and their cytokines provide critically needed predictive biomarkers for defining responders versus non- responders. IsoPlexis technology provides both the ability to rapidly isolate TILs from tumor samples and to detect their full functional cytokine excretion response to treatment. In Phase I of our IMAT SBIR Project, IsoPlexis developed a dual-function device that can capture a specific cell type from a complex mixture of cells via DNA tethered (DEAL) antibody technology developed in (SAB member, Cal Tech Professor) Jim Heath?s lab. Upon removal of the non-targeted cells, the captured T cells are then immediately released via site-specific DNA cleavage of the DEAL tether and transferred into a field of single-cell capture micro-troughs. Then these microtroughs are encapsulated by an antibody barcoded slide (SCBCTM). Once isolated in each microchamber, the barcode of antibodies collects 32+ different clinically relevant proteins being sent from each T cell. For Phase II of this project, we will further develop the duo cell capture technology integrating IsoPlexis automation technology to isolate melanoma TILs. Aim 1: Develop TILs capture and release system for immunotherapy and highly-multiplexed SCBC protein capture panel customized for TILs functional response profiling. Aim 2: Develop robust, fully automated assay processing for seamless incorporation into the clinical laboratory. Demonstrate the utility of the automation system for easy-to-use enrichment and analysis protocol validating with 30+ melanoma TILs samples. Aim 3: Implement fully automated workflow at Yale trial site to profile responders vs. non-responders across 20 patients. Combining technological improvements of Aims 1 and 2, IsoPlexis will demonstrate pre-and post-monitoring of responder differences, using multi-functional and phenotypic profile of cytotoxic T cells monitoring a mid-scale cohort of patients (20) over a 6 month period.